Cyclic GMP phosphodiesterase

ABSTRACT

The invention provides a human cyclic GMP phosphodiesterase (PDE9A) and polynucleotides which identify and encode PDE9A. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of PDE9A.

This application is a divisional application of U.S. application Ser.No. 09/240,359 filed Jan. 29, 1999, issued on July 3, 2001, as U.S. Pat.No. 6,255,456, entitled CYCLIC GMP PHOSPHODIESTERASE, which is adivisional application of U.S. application Ser. No. 08/987,466 filedDecember 9, 1997, issued on July 13, 1999, as U.S. Pat. No. 5,922,595,entitled CYCLIC OMP PHOSPHODIBSTJ3RASE, the contents of all which arehereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the nucleic acid and amino acid sequences of acyclic GMP phosphodiesterase and to the use of these sequences in thediagnosis, prevention, and treatment of cancer and immune disorders.

BACKGROUND OF THE INVENTION

Cyclic nucleotides (cAMP and cGMP) function as intracellular secondmessengers to transduce a variety of extracellular signals, includinghormones, light, and neurotransmitters. Cyclic nucleotidephosphodiesterases (PDEs) degrade cyclic nucleotides to thecorresponding monophosphates, thereby regulating the intracellularconcentrations of cyclic nucleotides and their effects on signaltransduction. At least seven families of mammalian PDEs have beenidentified based on substrate specificity and affinity, sensitivity tocofactors, and sensitivity to inhibitory drugs (Beavo, J. A. (1995)Physiological Reviews 75:725-48). Several of these families containdistinct genes many of which are expressed in different tissues assplice variants. Within families, there are multiple isozymes andmultiple splice variants of those isozymes. The existence of multiplePDE families, isozymes, and splice variants presents an opportunity forregulation of cyclic nucleotide levels and functions.

Type 1 PDEs (PDE1s) are Ca²⁺/calmodulin dependent, appear to containthree different genes, each having at least two different splicevariants. PDE1s have been found in the lung, heart, and brain. Some ofthe calmodulin-dependent PDEs are regulated in vitro byphosphorylation/dephosphorylation. Phosphorylation of PDE1 decreases theaffinity of the enzyme for calmodulin as well as PDE activity, whileincreasing steady state levels of cAMP. PDE2s are cGMP stimulated PDEsthat are localized in the brain that are thought to mediate the effectsof cAMP on catecholamine secretion. PDE3s are one of the major familiesof PDEs present in vascular smooth muscle. PDE3s are inhibited by cGMP,have high specificity for cAMP as a substrate, and play a role incardiac function. One isozyme of PDE3 is regulated by one or moreinsulin-dependent kinases. PDE4s are the predominant isoenzymes in mostinflammatory cells, some PDE4s are activated by cAMP-dependentphosphorylation. PDE5s are thought to be cGMP specific, but may alsoaffect cAMP function. High levels of PDE5s are found in most smoothmuscle preparations, in platelets and in the kidney. PDE6s play a rolein vision and are regulated by light and cGMP. The PDE7 class,consisting of only one known member, is cAMP specific and is mostclosely related to PDE4. PDE7 is not inhibited by rolipram, a specificinhibitor of PDE4 (See Beavo, supra). PDE8 represents a new family ofPDEs that are cAMP specific, most closely related to PDE4, insensitiveto rolipram, and sensitive to dipyridimole.

PDEs are composed of a catalytic domain of ˜270 amino acids, anN-terminal regulatory domain responsible for binding cofactors, and, insome cases, a C-terminal domain of unknown function. A conserved motif,HDXXHXGXXN, has been identified in the catalytic domain of all PDEs. PDEfamilies display approximately 30% amino acid identity within thiscatalytic domain, however isozymes within the same family typicallydisplay about 85-95% identity in this region (e.g. PDE4A vs PDE4B).Furthermore, within a family there is extensive similarity (>60%)outside the catalytic domain, while across families, there is little orno sequence similarity.

Many functions of immune and inflammatory responses are inhibited byagents that increase intracellular levels of cAMP (Verghese, M. W. etal. (1995) Mol. Pharmacol. 47:1164-1171). A variety of diseases havebeen attributed to increased PDE activity and associated with decreasedlevels of cyclic nucleotides. A form of diabetes insipidus in the mousehas been associated with increased PDE4 activity, and an increase inlow-K_(m) cAMP PDE activity has been reported in leukocytes of atopicpatients. Defects in PDEs have also been associated with retinaldisease. Retinal degeneration in the rd mouse, autosomal recessiveretinitis pigmentosa in humans, and rod/cone dysplasia 1 in Irish Setterdogs have been attributed to mutations in the PDE6B gene. PDE3 has beenassociated with cardiac disease.

Many inhibitors of PDEs have been identified and have undergone clinicalevaluation. PDE3 inhibitors are being developed as antithromboticagents, antihypertensive agents, and as cardiotonic agents useful in thetreatment of congestive heart failure. Rolipram, a PDE4 inhibitor, hasbeen used in the treatment of depression, and other inhibitors of PDE4are undergoing evaluation as anti-inflammatory agents. Rolipram has alsobeen shown to inhibit lipopolysaccharide (LPS) induced TNF-alpha whichhas been shown to enhance HIV-1 replication in vitro. Therefore,rolipram may inhibit HIV-1 replication (Angel, J. B. et al. (1995) AIDS9:1137-44). Additionally, rolipram, based on its ability to suppress theproduction of cytokines such as TNF alpha and beta and interferon gamma,has been shown to be effective in the treatment of encephalomyelitis.Rolipram may also be effective in treating tardive dyskinesia and waseffective in treating multiple sclerosis in an experimental animal model(Sommer, N. et al. (1995) Nat. Med. 1:244-248; Sasaki, H. et al. (1995)Eur. J. Pharmacol 282:71-76).

Theophylline is a nonspecific PDE inhibitor used in the treatment ofbronchial asthma and other respiratory diseases. Theophylline isbelieved to act on airway smooth muscle function and in ananti-inflammatory or immunomodulatory capacity in the treatment ofrespiratory diseases (Banner, K. H. and Page, C. P. (1995) Eur. Respir.J. 8:996-1000). Pentoxifylline is another nonspecific PDE inhibitor usedin the treatment of intermittent claudication and diabetes-inducedperipheral vascular disease. Pentoxifylline is also known to blockTNF-alpha production and may inhibit HIV-1 replication (Angel et al.,supra).

PDEs have also been reported to effect cellular proliferation of avariety of cell types and have been implicated in various cancers. Banget al. (1994; Proc Natl Acad Sci USA 91:5330-5334) reported that growthof prostate carcinoma cell lines DU 145 and LNCaP was inhibited bydelivery of cAMP derivatives and phosphodiesterase inhibitors. Thesecells also showed a permanent conversion in phenotype from epithelial toneuronal morphology. Others have suggested that PDE inhibitors have thepotential to regulate mesangial cell proliferation and lymphocyteproliferation (Matousovic, K. et al. (1995) J. Clin. Invest. 96:401-410;Joulain, C. et al. (1995) J. Lipid Mediat. Cell Signal. 11:63-79,respectively). Finally, Deonarain et al. (1994; Br. J. Cancer 70:786-94)describe a cancer treatment that involves intracellular delivery ofphosphodiesterases to particular cellular compartments of tumors whichresults in cell death.

The discovery of new cyclic nucleotide phosphodiesterases and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention, andtreatment of cancer and immune disorders.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, cyclic GMPphosphodiesterase (PDE9A), comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified variant of PDE9Ahaving at least 90% amino acid identity to the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1. The invention also includes an isolated and purifiedpolynucleotide variant having at least 90% polynucleotide identity tothe polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

Additionally, the invention provides a composition comprising apolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionfurther provides an isolated and purified polynucleotide sequence whichhybridizes under stringent conditions to the polynucleotide sequenceencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purifiedpolynucleotide sequence which is complementary to the polynucleotidesequence encoding the polypeptide comprising the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidesequence comprising SEQ ID NO:2 or a fragment of SEQ ID NO:2, and anisolated and purified polynucleotide variant having at least 90%polynucleotide identity to the polynucleotide sequence comprising SEQ IDNO:2 or a fragment of SEQ ID NO:2. The invention also provides anisolated and purified polynucleotide sequence which is complementary tothe polynucleotide sequence comprising SEQ ID NO:2 or a fragment of SEQID NO:2.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1. In another aspect, the expression vector is contained within ahost cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcontaining an expression vector containing at least a fragment of apolynucleotide sequence encoding PDE9A under conditions suitable for theexpression of the polypeptide; and (b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified PDE9A having the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1, as well as a purified agonist and a purifiedantagonist of the polypeptide.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of PDE9A.

The invention also provides a method for treating or preventing animmune disorder, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of PDE9A.

The invention also provides a method for detecting a polynucleotideencoding PDE9A in a biological sample containing nucleic acids, themethod comprising the steps of: (a) hybridizing the complement of thepolynucleotide sequence encoding the polypeptide comprising SEQ ID NO:1or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of thebiological sample, thereby forming a hybridization complex; and (b)detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotideencoding PDE9A in the biological sample. In one aspect, the nucleicacids of the biological sample are amplified by the polymerase chainreaction prior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show the amino acid sequence (SEQ IDNO:1) and nucleic acid sequence (SEQ ID NO:2) of PDE9A. The alignmentswere produced using MACDNASIS PRO software (Hitachi Software EngineeringCo. Ltd. San Bruno, Calif.).

FIGS. 2A, 2B, 2C, and 2D, show the amino acid sequence alignments amongPDE9A (828228; SEQ ID NO:1), PDE8A (SEQ ID NO:3), and a cAMP-specificPDE from Drosophila melanogaster (GI 829179; SEQ ID NO:4), producedusing the multisequence alignment program of DNASTAR software (DNASTARInc, Madison Wis.).

FIG. 3 shows the double-reciprocal, Lineweaver-Burke plot for theactivity of PDE9A using cGMP as a substrate; the positive X axisreflects the reciprocal of the substrate (cGMP) concentration (1/S), andthe positive Y axis reflects the reciprocal of the reaction velocity(1/V). Lineweaver-Burke analysis was performed according to Segal, I. H.(Enzyme Kinetics (1995) pp. 214-245, John Wiley and Sons, New York,N.Y.)

FIG. 4 shows the dependence of PDE9A activity on divalent cationconcentration; the positive X axis reflects cation concentration (mM),and the positive Y axis reflects the percent hydrolysis of cAMP.Divalent cations tested were calcium chloride (CaCL₂; squares),magnesium chloride (MgCl₂; open circles), and manganese chloride (MnCl₂;closed circles).

FIG. 5 shows the effect of various PDE inhibitors on the activity ofPDE9A; the positive X axis reflects the concentration of inhibitor (M),and the positive Y axis reflects the percent hydrolysis of cGMP relativeto an uninhibited control incubation (100%).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein, and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

PDE9A, as used herein, refers to the amino acid sequences ofsubstantially purified PDE9A obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, whenbound to PDE9A, increases or prolongs the duration of the effect ofPDE9A. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of PDE9A.

An “allele” or “allelic sequence,” as used herein, is an alternativeform of the gene encoding PDE9A. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding PDE9A as used herein, includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent PDE9A. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding PDE9A,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding PDE9A. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent PDE9A.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of PDE9A is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

“Amino acid sequence,” as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of PDE9A arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of PDE9A. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

“Amplification,” as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term “antagonist,” as used herein, refers to a molecule which, whenbound to PDE9A, decreases the amount or the duration of the effect ofthe biological or immunological activity of PDE9A. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of PDE9A.

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fab, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind PDE9Apolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term “antisense,” as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

The term “biologically active,” as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic PDE9A, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A “composition comprising a given polynucleotide sequence,” as usedherein, refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding PDE9A may be employed as hybridization probes. The probes maybe stored in freeze-dried form and may be associated with a stabilizingagent such as a carbohydrate. In hybridizations, the probe may bedeployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The phrase “consensus sequence,” as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW Fragment Assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding PDE9A, bynorthern analysis is indicative of the presence of nucleic acidsencoding PDE9A in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding PDE9A.

A “deletion,” as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to PDE9A or theencoded PDE9A. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword “identity” may substitute for the word “homology.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term “humanized antibody,” as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term “hybridization,” as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term “hybridization complex,” as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

The term “microarray,” as used herein, refers to an array of distinctpolynucleotides or oligonucleotides arrayed on a substrate, such aspaper, nylon or any other type of membrane, filter, chip, glass slide,or any other suitable solid support.

The term “modulate,” as used herein, refers to a change in the activityof PDE9A. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of PDE9A.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, “fragments” refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms“amplimers”, “primers”, “oligomers”, and “probes”, as commonly definedin the art.

“Peptide nucleic acid”, PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term “sample,” as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding PDE9A,or fragments thereof, or PDE9A itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like.

The terms “specific binding” or “specifically binding,” as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope “A”, the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled “A” and the antibody will reduce the amount of labeled A boundto the antibody.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt and/or formamide inthe prehybridization and hybridization solutions, or by thehybridization temperature, and are well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5X SSPE, 0.3%SDS, and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide or/and at a reduced temperature of 35° C. Thetemperature range corresponding to a particular level of stringency canbe further narrowed by calculating the purine to pyrimidine ratio of thenucleic acid of interest and adjusting the temperature accordingly.Variations on the above ranges and conditions are well known in the art.

The term “substantially purified,” as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A “variant” of PDE9A, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software.

The Invention

The invention is based on the discovery of a new human cyclic-GMPspecific phosphodiesterase (PDE9A), the polynucleotides encoding PDE9A,and the use of these compositions for the diagnosis, prevention, ortreatment of cancer and immune disorders.

Nucleic acids encoding the PDE9A of the present invention were firstidentified in Incyte Clone 828228 from the prostate tissue cDNA library(PROSNOT06) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from extension of thenucleic acid sequence of this clone.

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,1D, 1E, and 1F. PDE9A is 593 amino acids in length and has a consensussignature sequence for cyclic nucleotide PDEs at H₃₅₂DLDHPGYNN. Thissequence is a part of one of two potential divalent cation binding sitesconserved in PDEs, and having the general structure of HXXXH(X₆₋₂₄)E.The first of these sites is found in the sequence H₃₁₂ - - - H₃₁₆ - - -D₃₄₁, and has D₃₄₁ as a conservative amino acid substitution for E. Thissubstitution is found in at least one other PDE, PDE7. The second ofthese sites is found in the sequence H₃₅₂ - - - H₃₅₆ - - - E₃₈₂. Asshown in FIGS. 2A, 2B, 2C, and 2, PDE9A has chemical and structuralhomology with PDE8A (SEQ ID NO:3) and the cAMP-specific PDE from D.melanogaster (GI 829179; SEQ ID NO:4). In particular, PDE9A shares 24%identity with PDE8A and 20% identity with D. melanogaster cAMP PDE. The˜270 amino acid catalytic domain found in all PDEs extends approximatelybetween residues F₂₈₈ and W₅₄₄ for PDE9A, and is 34% identical to PDE8Aand 30% identical to D. melanogaster PDE in this region. The threeproteins share the two divalent cation binding sites and the consensussignature sequence, HDXXHXGXXN. PDE9A exhibits a similar degree ofhomology (28% to 32%) in the catalytic domain to other representativesof the PDE families 1, 2, 3, 4, 5, 6, and 7 (data not shown).

A 1.8 kb region of PDE9A encoding the full length of the protein wascloned into the baculovirus transfer vector pFASTBAC, expressed in sf9cells, and a cell lysate prepared from these cells for enzyme assays.FIG. 3 shows the kinetics of enzyme activity of recombinant, purifiedPDE9A with cGMP as a substrate. PDE9A has a very high affinity for cGMPwith a Km of 170 nM, and a very low affinity for cAMP (Km=230 υM, datanot shown). FIG. 4 shows the dependence of PDE9A on divalent cations formaximal activity with a preference for Mn⁺⁺ over Mg⁺⁺ or Ca⁺⁺. Theeffects of various known PDE inhibitors on the activity of PDE9A areshown in FIG. 5. PDE9A was not inhibited by up to 100 μM of rolipram(inhibitor of PDE4), dipyridamole (inhibitor of PDE2, 4, 5, and 6),SKF94120 (inhibitor of PDE3), vinpocetine (inhibitor of PDE1), or IBMX(non-specific PDE inhibitor). PDE9A was inhibited by zaprinast(inhibitor of PDE5 and 6) with an IC₅₀ of 35 μM. Membrane-based northernanalysis shows the expression of this sequence in various tissues, withthe most significant expression in testis, ovary, small intestine, andcolon. Electronic northern analysis using the LIFESEQ database furthershows the expression of this sequence in various tissues, at least 50%of which are cancerous and at least 25% of which involves inflammationor the immune response. Of particular note is the expression of PDE9A inCrohn's disease.

The degree of similarity exhibited between the PDE9A and representativesof the other eight families of PDEs (28% to 30%) is consistent with thatdemonstrated between different PDE families (˜30%). PDE9A is furtherdistinguished from other known families by its specificity for cGMP andpattern of inhibition by known PDE inhibitors. PDE9A therefore appearsto be a member of a new family of cyclic nucleotide phosphodiesterasesdesignated PDE9.

The invention also encompasses PDE9A variants. A preferred PDE9A variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe PDE9A amino acid sequence, and which contains at least onefunctional or structural characteristic of PDE9A.

The invention also encompasses polynucleotides which encode PDE9A. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes a PDE9A.

The invention also encompasses a variant of a polynucleotide sequenceencoding PDE9A. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding PDE9A. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding PDE9A, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring PDE9A, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode PDE9A and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring PDE9A under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding PDE9A or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding PDE9A and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode PDE9A and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding PDE9A or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency as taught in Wahl, G. M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987;Methods Enzymol. 152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by GIBCO/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding PDE9A may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTER FINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode PDE9A may be used in recombinant DNAmolecules to direct expression of PDE9A, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressPDE9A.

As will be understood by those of skill in the art, it may beadvantageous to produce PDE9A-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter PDE9Aencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding PDE9A may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of PDE9A activity, it may be useful toencode a chimeric PDE9A protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the PDE9A encoding sequence and theheterologous protein sequence, so that PDE9A may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding PDE9A may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of PDE9A, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of PDE9A, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active PDE9A, the nucleotidesequences encoding PDE9A or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding PDE9A andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding PDE9A. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector-enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (GIBCO/BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding PDE9A,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for PDE9A. For example, when largequantities of PDE9A are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding PDE9A may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding PDE9A may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

An insect system may also be used to express PDE9A. For example, a 1.8kb region of PDE9A encoding the full length protein was PCR-amplifiedand cloned into the baculovirus transfer vector pFASTBAC (LifeTechnologies, Inc., Gaithersburg, Md.), which had been modified toinclude a 5′ FLAG tag. Recombinant virus stocks were prepared accordingto the manufacturer's protocol. Sf9 cells were cultured in Sf900 II Sfmserum free media (Life Technologies Inc.) at 27° C. For expression,1×10⁸ Sf9 cells were infected at a multiplicity of infection of 5 in afinal volume of 50 mls. Three days post-infection, the cells wereharvested and enzyme-containing lysates were prepared. To monitorexpression, 1 μl each of mock-infected and PDE9A-infected cell lysatewas electrophoresed in a polyacrylamide gel and either silver-stained bystandard methods or transferred to nitrocellulose and Western blottedwith an anti-FLAG antibody (M2, Scientific Imaging System, EastmanKodak, New Haven, Conn.) at a concentration of 2 mg/ml.

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding PDE9A may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing PDE9A in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding PDE9A. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding PDE9A, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressPDE9A may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding PDE9A isinserted within a marker gene sequence, transformed cells containingsequences encoding PDE9A can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding PDE9A under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding PDE9A and express PDE9A may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding PDE9A can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding PDE9A. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding PDE9A to detect transformantscontaining DNA or RNA encoding PDE9A.

A variety of protocols for detecting and measuring the expression ofPDE9A, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson PDE9A is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding PDE9A includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding PDE9A, orany fragments thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison, Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio)).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding PDE9A may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodePDE9A may be designed to contain signal sequences which direct secretionof PDE9A through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding PDE9A to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and PDE9A may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingPDE9A and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying PDE9A from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of PDE9A may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield, J. (1963) J. Am. Chem. Soc. 85:2149-2154). Proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments ofPDE9A may be chemically synthesized separately and combined usingchemical methods to produce the full length molecule.

Therapeutics

Chemical and structural homology exists among PDE9A, and PDE8A, and D.melanogaster cAMP PDE. In addition, PDE9A is expressed in cancer andtissues associated with inflammation and the immune response. Therefore,PDE9A appears to play a role in cancer and immune disorders. Inparticular, inhibitors of PDE have been shown to be effective in thetreatment of these types of diseases and disorders.

Therefore, in one embodiment, an antagonist of PDE9A may be administeredto a subject to prevent or treat a cancer. Such cancers may be, but arenot limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds PDE9A may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express PDE9A.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding PDE9A may be administered to a subject to treator prevent a cancer including, but not limited to, the types of cancerdescribed above.

In another embodiment, an antagonist of PDE9A may be administered to asubject to prevent or treat an immune disorder. Such disorders mayinclude, but are not limited to, AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding PDE9A may be administered to a subject to treator prevent an immune disorder including, but not limited to, thosedescribed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of PDE9A may be produced using methods which are generallyknown in the art. In particular, purified PDE9A may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind PDE9A.

Antibodies to PDE9A may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith PDE9A or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to PDE9A have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of PDE9A amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to PDE9A may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producePDE9A-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton, D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for PDE9A mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between PDE9A and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering PDE9A epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingPDE9A, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding PDE9A may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding PDE9A. Thus, complementary molecules orfragments may be used to modulate PDE9A activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding PDE9A.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingPDE9A. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding PDE9A can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes PDE9A. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′ or regulatory regions of the gene encoding PDE9A(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions−10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using “triple helix” base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding PDE9A.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding PDE9A. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of PDE9A, antibodies toPDE9A, mimetics, agonists, antagonists, or inhibitors of PDE9A. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of PDE9A, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example PDE9A or fragments thereof, antibodies of PDE9A,agonists, antagonists or inhibitors of PDE9A, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind PDE9A may beused for the diagnosis of conditions or diseases characterized byexpression of PDE9A, or in assays to monitor patients being treated withPDE9A, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for PDE9A includemethods which utilize the antibody and a label to detect PDE9A in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringPDE9A are known in the art and provide a basis for diagnosing altered orabnormal levels of PDE9A expression. Normal or standard values for PDE9Aexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toPDE9A under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of PDE9A expressed insubject, control, and disease, samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingPDE9A may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofPDE9A may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofPDE9A, and to monitor regulation of PDE9A levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding PDE9A or closely related molecules, may be used to identifynucleic acid sequences which encode PDE9A. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding PDE9A, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe PDE9A encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring PDE9A.

Means for producing specific hybridization probes for DNAs encodingPDE9A include the cloning of nucleic acid sequences encoding PDE9A orPDE9A derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding PDE9A may be used for the diagnosis ofconditions or disorders which are associated with expression of PDE9A.Examples of such conditions or disorders include, but are not limitedto, cancers, such as adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, and teratocarcinoma, and, in particular, cancers ofthe adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus; and immunedisorders, such as AIDS, Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, gout, Graves' disease,hypereosinophilia, irritable bowel syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, myocardial or pericardialinflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis,rheumatoid arthritis, scleroderma, Sjögren's syndrome, and autoimmunethyroiditis; complications of cancer, hemodialysis, extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections; and trauma. The polynucleotide sequences encodingPDE9A may be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered PDE9A expression. Such qualitative orquantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding PDE9A may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingPDE9A may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding PDE9A in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of PDE9A, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes PDE9A, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding PDE9A may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5→3′) and another with antisense (3′←5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of PDE9Ainclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously (to produce atranscript image), and to identify genetic variants, mutations andpolymorphisms. This information may be used to determine gene function,to understand the genetic basis of disease, to diagnose disease, and todevelop and monitor the activities of therapeutic agents.

In one embodiment, the microarray is prepared and used according to themethods known in the art such as those described in PCT applicationWO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech.14:1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci.93:10614-10619).

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably about 15 to 30 nucleotides in length, and mostpreferably about 20 to 25 nucleotides in length. For a certain type ofmicroarray, it may be preferable to use oligonucleotides which are only7 to 10 nucleotides in length. The microarray may containoligonucleotides which cover the known 5′ (or 3′) sequence, or maycontain sequential oligonucleotides which cover the full lengthsequence; or unique oligonucleotides selected from particular areasalong the length of the sequence. Polynucleotides used in the microarraymay be oligonucleotides that are specific to a gene or genes of interestin which at least a fragment of the sequence is known or that arespecific to one or more unidentified cDNAs which are common to aparticular cell or tissue type or to a normal, developmental, or diseasestate. In certain situations, it may be appropriate to use pairs ofoligonucleotides on a microarray. The pairs will be identical, exceptfor one nucleotide preferably located in the center of the sequence. Thesecond oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from 2 to1,000,000.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In one aspect, the oligomers aresynthesized at designated areas on a substrate using a light-directedchemical process. The substrate may be paper, nylon or any other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

In one aspect, the oligonucleotides may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jetapplication apparatus, such as that described in PCT applicationWO95/251116 (Baldeschweiler et al.). In another aspect, a “gridded”array analogous to a dot or slot blot (HYBRIDOT apparatus, GIBCO/BRL)may be used to arrange and link cDNA fragments or oligonucleotides tothe surface of a substrate using a vacuum system, thermal, UV,mechanical or chemical bonding procedures. In yet another aspect, anarray may be produced by hand or by using available devices, materials,and machines (including BRINKMANN multichannel pipettors or roboticinstruments) and may contain 8, 24, 96, 384, 1536 or 6144oligonucleotides, or any other multiple from 2 to 1,000,000 which lendsitself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a biological sample. The biologicalsamples may be obtained from any bodily fluid (blood, urine, saliva,phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissuepreparations. To produce probes, the polynucleotides extracted from thesample are used to produce nucleic acid sequences which arecomplementary to the nucleic acids on the microarray. If the microarrayconsists of cDNAs, antisense RNAs (aRNA) are appropriate probes.Therefore, in one aspect, mRNA is used to produce cDNA which, in turnand in the presence of fluorescent nucleotides, is used to producefragment or oligonucleotide aRNA probes. These fluorescently labeledprobes are incubated with the microarray so that the probe sequenceshybridize to the cDNA oligonucleotides of the microarray. In anotheraspect, nucleic acid sequences used as probes can includepolynucleotides, fragments, and complementary or antisense sequencesproduced using restriction enzymes, PCR technologies, and Oligolabelingor TransProbe kits (Pharmacia) well known in the area of hybridizationtechnology.

Incubation conditions are adjusted so that hybridization occurs withprecise complementary matches or with various degrees of lesscomplementarity. After removal of nonhybridized probes, a scanner isused to determine the levels and patterns of fluorescence. The scannedimages are examined to determine degree of complementarity and therelative abundance of each oligonucleotide sequence on the microarray. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies or functional analysis of the sequences, mutations, variants, orpolymorphisms among samples (Heller, R. A. et al., (1997) Proc. Natl.Acad. Sci. 94:2150-55).

In another embodiment of the invention, the nucleic acid sequences whichencode PDE9A may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingPDE9A on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, PDE9A, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenPDE9A and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to PDE9A large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with PDE9A, or fragments thereof, and washed. Bound PDE9A isthen detected by methods well known in the art. Purified PDE9A can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding PDE9A specificallycompete with a test compound for binding PDE9A. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with PDE9A.

In additional embodiments, the nucleotide sequences which encode PDE9Amay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I PROSNOT06 cDNA Library Construction

The PROSNOT06 cDNA library was constructed from microscopically normalprostate tissue obtained from a 57-year-old Causcasian male. This tissuewas associated with cDNA library PROSTUT04, a prostate tumor from thesame patient. Both tissues were excised when the patient during aradical prostatectomy which included removal of both testes and excisionof regional lymph nodes. Pathology indicated adenofibromatoushyperplasia and adenocarcinoma (Gleason grade 3+3) in both the right andleft periphery of the prostate. There was perineural invasion, and thetumor perforated the capsule. The patient history reported a benignneoplasm of the large bowel. The patient was taking insulin for type Idiabetes. The patient's family history included a malignant neoplasm ofthe prostate in the father and type I diabetes without complications inthe mother.

The frozen tissue was homogenized and lysed using a Brinkman HomogenizerPolytron-PT 3000 (Brinkman Instruments, Inc. Westbury, N.Y.) inguanidinium isothiocyanate solution. 1.0 ml of 2M sodium acetate wasadded to the lysate and the lysate was extracted once with phenolchloroform at pH 5.5 per Stratagene's RNA isolation protocol(Stratagene), and once with acid phenol at pH 4.7. The RNA wasprecipitated with an equal volume of isopropanol according toStratagene's protocol. RNA pellet was resuspended in DEPC-treated waterand treated with DNase for 50 min at 37 C. The reaction was stopped withan equal volume of acid phenol, and the RNA was precipitated using 0.3 Msodium acetate and 2.5 volumes of ethanol and resuspended inDEPC-treated water. The RNA was isolated with the OLIGOTEX kit (QIAGENInc, Chatsworth, Calif.) and used to construct the cDNA library.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT Plasmid System for cDNA synthesis and plasmid cloning (Cat.#18248-013; GIBCO/BRL, Gaithersburg, Md.). cDNAs were fractionated on aSEPHAROSE CL4B colmn (Cat. 275105, Pharmacia), and those cDNAs exceeding400 bp were ligated into PSPORT1. The plasmid PSPORT1 was subsequentlytransformed into DH5α competent cells (Cat. #18258-012, GIBCO/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit (Catalog #26173, QIAGEN). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedwith the following modifications: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, GIBCO/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems; andthe reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol 36:290-300; Altschul, et al. (1990)J. Mol. Biol. 215:403-410).

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith, T. et al. (1992, Protein Engineering5:35-51), incorporated herein by reference, could have been used whendealing with primary sequence patterns and secondary structure gappenalties. The sequences disclosed in this application have lengths ofat least 49 nucleotides, and no more than 12% uncalled bases (where N isrecorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻¹⁰ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam);and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Human multiple tissue northern blots (Clontech, Palo Alto, Calif.) werehybridized with a probe consisting of the 5′ most 1090 nucleotides ofclone 828228. Probe DNA was labeled with ³²P using the “Ready-To-Go”random prime labeling kit (Pharmacia Biotech Inc., Piscataway, N.J.) andwashed to a stringency of 0.5×SSC, 65° C. The highest levels of PDE9Awere seen in spleen, small intestine, and brain, but detectable levelswere seen in all tissues examined.

Computer techniques analogous to membrane based northern analysis werealso performed using BLAST (Altschul (1993), supra; Altschul (1990),supra). The basis of the search is the product score which is definedas:% sequence identity×% maximum BLAST score/100The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding PDE9A occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of PDE9A Encoding Polynucleotides

cDNA sequences were extended by PCR amplification using human λgt10testis or stomach cDNA libraries (Clontech Laboratories, Inc. Palo Alto,Calif.) and nested primers. For each reaction, 2.5×10⁷ pfu were boiledfor 5 minutes to release DNA. First round PCR (15 cycles) was performedwith a PDE9A specific primer (9A specific-outer:5′-GGGTGACAGGGTTGATGCT-3′; SEQ ID NO:5) and either a λgt10 forward(5′-TCGCTTAGTTTTACCGTTTTC-3′ (SEQ ID NO:6), or a λgt10 reverse(5′-TATCGCCTCCATCAACAAACTT-3′; SEQ ID NO:7) primer. An aliquot,{fraction (1/50)} of the reaction mixture, was used as a template for asecond round of amplification (30 cycles) with a PDE9A specific primer(9A specific-inner: 5′-GACACAGAACAGCCACCTC-3′; SEQ ID NO:8) with eithera nested λgt10 forward (5′-AGCAAGTTCAGCCTGGTTAAG-3′; SEQ ID NO:9) orλgt10 reverse (5′-CTTATGAGTATTTCTTCCAGGGTA-3′; SEQ ID NO:10) primer.Southern analysis of the PCR products used an internal PDE9Ahybridization probe (5′-ATCATGGTTACAAATTATCGAAGCCAATTA-3′; SEQ IDNO:11). 5′ RACE amplification was also performed on human brain mRNA(Clontech) to extend the sequence. 5′ RACE was performed using a “5′RACE System for Rapid Amplification of cDNA Ends” kit (LifeTechnologies, Inc., Grand Island, N.Y.) according to the manufacturer'sprotocol. PDE9A specific primers used in the 5′ RACE were: ReverseTranscriptase primer, 5′-GCTCCTCCCTCATCTTCTTA-3′ (SEQ ID NO:12); Outerprimer, 5′-AGGACAGCCAAGTGATTT-3′ (SEQ ID NO:13); Inner primer,5′-TGCGCTGGCCTTCCTGGTAG-3′ (SEQ ID NO:14). Positive bands were subclonedand sequenced. All sequences subsequently incorporated into the extendedPDE9A sequence were verified by sequencing multiple independent PCRamplifications from the cDNA library DNA using unique primers or byindependent amplification from mRNA.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from polynucleotide sequences of theinvention are employed to screen cDNAs, genomic DNAs, or mRNAs. Althoughthe labeling of oligonucleotides, consisting of about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger nucleotide fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO 4.06 (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-³²P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn).A aliquot containing 10⁷ counts per minute of the labeled probe is usedin a typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases (Ase I, Bgl II, Eco RI,Pst I, XbaI, or Pvu II; DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots in a PHOSPHOIMAGERcassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, one of the nucleotidesequences of the present invention is examined using a computeralgorithm which starts at the 3′ end of the nucleotide sequence. Thealgorithm identified oligomers of defined length that are unique to thegene, have a GC content within a range suitable for hybridization, andlack predicted secondary structure that would interfere withhybridization. The algorithm identifies approximately 20sequence-specific oligonucleotides of 20 nucleotides in length(20-mers). A matched set of oligonucleotides are created in which onenucleotide in the center of each sequence is altered. This process isrepeated for each gene in the microarray, and double sets of twenty 20mers are synthesized and arranged on the surface of the silicon chipusing a light-directed chemical process, such as that discussed in Chee,supra.

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate (cf.Baldeschweiler, supra). In another alternative, a “gridded” arrayanalogous to a dot (or slot) blot is used to arrange and link cDNAfragments or oligonucleotides to the surface of a substrate using avacuum system, thermal, UV, mechanical or chemical bonding procedures. Atypical array may be produced by hand or using available materials andmachines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536dots or 6144 dots. After hybridization, the microarray is washed toremove nonhybridized probes, and a scanner is used to determine thelevels and patterns of fluorescence. The scanned image is examined todetermine degree of complementarity and the relativeabundance/expression level of each oligonucleotide sequence in themicroarray.

VIII Complementary Polynucleotides

Sequences complementary to the PDE9A-encoding sequence, or any partthereof, are used to decrease or inhibit expression of naturallyoccurring PDE9A. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of PDE9A. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the PDE9A-encoding transcript.

IX Expression of PDE9A

A 1.8 kb region of PDE9A encoding the full length protein (nucleotides61-1842) was amplified and cloned into the baculovirus transfer vectorPFASTBAC (Life Technologies, Inc., Gaithersburg, Md.), which had beenmodified to include a 5′ FLAG tag. Recombinant virus stocks wereprepared according to the manufacturer's protocol. Sf9 cells werecultured in Sf900 II Sfm serum free media (Life Technologies Inc.) at27° C. For expression, 1×10⁸ Sf9 cells were infected at a multiplicityof infection of 5 in a final volume of 50 mls. At three dayspost-infection, the cells were harvested, and enzyme-containing lysateswere prepared as detailed below. To monitor expression, 1 ml each ofmock-infected and PDE9A infected cell lysate was electrophoresed in apolyacrylamide gel and either silver-stained by standard methods ortransferred to nitrocellulose and assayed using western analysis and ananti-FLAG antibody (M2, Scientific Imaging System, Eastman Kodak, NewHaven, Conn.) at a concentration of 2 mg/ml. The secondary antibody wasan alkaline phosphatase conjugated anit-mouse IgG (Boehringer Mannheim,Indianapolis, Ind.) and the blot was visualized with a “BCIP/NBTphosphatase substrate system” (Kirkegaard & Perry Laboratories,Gaithersburg, Md.) according to the manufactur's protocol.

PDE9A to be used for assay was prepared from transfected Sf9 cells.Cells were harvested by centrifugation, resuspended in homogenizationbuffer (20 mM Tris-HCl, 2 mM benzamidine, 1 mM EDTA, 0.25 M sucrose, 100uM PMSF, pH 7.5 ) at 1×10⁷ cells/ml, and disrupted using a Bransonsonicating probe (3×10 second pulses). Cellular debris was removed bycentrifugation at 14,000×g for 10 minutes. The supernatant was stored at−70° C.

X Demonstration of PDE9A Activity

PDE activity was assayed by measuring the conversion of ³H-cGMP to³H-guanosine in the presence of PDE9A and 5′ nucleotidase. A one-stepassay was run using a 100 uL assay containing 50 mM Tris-HCl pH 7.5, 10mM MgCl², 0.1 unit 5′ nucleotidase (from Crotalus atrox venom), and0.0064-2.0 uM ³H-cGMP. The reaction was started by the addition of 25 μlof diluted enzyme supernatant. Reactions were run directly in miniPoly-Q scintillation vials (Beckman Instruments Inc., Fullerton Calif.).Assays were incubated at 37° C. for a time period that would yield lessthan 15% cGMP hydrolysis in order to avoid non-linearity associated withproduct inhibition. The reaction was stopped by the addition of 1 ml ofDowex AG1x8 (Cl form) resin (1:3 slurry). Three ml of scintillationfluid were added, and the vials were mixed. The resin in the vials wasallowed to settle for 1 hr before counting. Soluble radioactivityassociated with ³H-guanosine was quantitated using a Beta scintillationcounter. The amount of radioactivity recovered is proportional to theactivity of PDE9A in the reaction.

XI Production of PDE9A Specific Antibodies

PDE9A that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using LASERGENEsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

XII Purification of Naturally Occurring PDE9A Using Specific Antibodies

Naturally occurring or recombinant PDE9A is substantially purified byimmunoaffinity chromatography using antibodies specific for PDE9A. Animmunoaffinity column is constructed by covalently coupling PDE9Aantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing PDE9A is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof PDE9A (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/PDE9A binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and PDE9A iscollected.

XIII Identification of Molecules Which Interact with PDE9A

PDE9A or biologically active fragments thereof are labeled with ¹²⁵IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133:529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled PDE9A, washed and any wells withlabeled PDE9A complex are assayed. Data obtained using differentconcentrations of PDE9A are used to calculate values for the number,affinity, and association of PDE9A with the candidate molecules.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. An isolated polynucleotide encoding a polypeptide selected from thegroup consisting of: a) a polypeptide comprising the amino acid sequenceof SEQ ID NO:1, b) a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence of SEQ ID NO:1, c) afragment of a polypeptide having the amino acid sequence of SEQ 1) NO:1,said fragment having cyclic nucleotide phosphodiesterase activity, andd) an immunogenic fragment of a polypeptide of at least 5 amino acids ofthe amino acid sequence of SEQ ID NO:1, said immunogenic fragment isused to make an antibody which specifically binds to an isolatedpolypeptide selected from the group consisting of a), b) and c).
 2. Arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 1. 3. A cell transformed with arecombinant polynucleotide of claim
 2. 4. A method for producing apolypeptide encoded by the polynucleotide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide of claim 1, and b)recovering the polypeptide so expressed.
 5. A method of claim 4, whereinthe polypeptide comprises the amino acid sequence of SEQ ID NO:1.
 6. Anisolated polynucleotide selected from the group consisting of: a) apolynucleotide comprising the polynucleotide sequence of SEQ II) NQ:2,b) a polynucleotide comprising a polynucleotide sequence at least 90%identical to the polynucleotide sequence of SEQ ID NO:2, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complemeiltary to a polynucleotide of b) and e) an RNAequivalent of a)-d).
 7. A method for detecting a target polynucleotidein a sample, said target polynucleotide having a sequence of apolynucleotide of claim 6, the method comprising: a) hybridizing thesample with a probe comprising at least 20 contiguous nucleotidescomprising a sequence complementary to said target polynucleotide in thesample, and which probe specifically hybridizes to said targetpolynucleotide, under conditions whereby a hybridization complex isformed between said probe and said target polynucleotide or fragmentsthereof, and b) detecting the presence or absence of said hybridizationcomplex, and, optionally, if present, the amount thereof.
 8. A method ofclaim 7, wherein the probe comprises at least 60 contiguous nucleotides.9. A method for detecting a target polynucleotide sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 6, themethod comprising: a) amplifying said target polynucleotide or fragmentthereof using polymerase chain reaction amplification, and b) detectingthe presence or absence of said amplified target polynucleotide orfragment thereof, and, optionally, if present, the amount thereof.
 10. Amethod for assessing toxicity of a test compound, said methodcomprising: a) treating a biological sample containing nucleic acidswith the test compound; b) hybridizing the nucleic acids of the treatedbiological sample with a probe comprising at least 20 contiguousnucleotides of a polynucleotide of claim 6 under conditions whereby aspecific hybridization complex is formed between said probe and a targetpolynucleotide in the biological sample, said target polynucleotidecomprising a polynucleotide sequence of a polynucleotide of claim 6 orfragment thereof; c) quantifying the amount of hybridization complex;and d) comparing the amount of hybridization complex in the treatedbiological sample with the amount of hybridization complex in anuntreated biological sample, wherein a difference in the amount ofhybridization complex in the treated biological sample is indicative oftoxicity of the test compound.
 11. A polynucleotide of claim 6,comprising the polynucleotide sequence of SEQ ID NO:2.
 12. A method forscreening a compound for effectiveness in altering expression of atarget polynucleotide, wherein said target polynucleotide comprises apolynucleotide sequence of claim 11, the method comprising: a) exposinga sample comprising the target polynucleotide to a compound, underconditions suitable for the expression of the target polynucleotide, b)detecting altered expression of the target polynucleotide, and c)comparing the expression of the target polynucleotide in the presence ofvarying amounts of the compound and in the absence of the compound.